With the completion of the human genome project, it has become clear that the number of genes cannot account for the complexity of the human proteome. This conclusion has lead to a dramatic increase in our appreciation of the abundance and importance of post-transcriptional mechanisms of gene regulation. Among several proposed mechanisms, alternative pre-mRNA splicing is considered to be one of the most efficient and wide spread avenues to generate multiple protein isoforms from individual genes. Current estimates indicate that over 90% of human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. This renewal application focuses on understanding the mechanisms of splice site selection with the long-term goal to generate a splicing code that permits alternative splicing predictions based on sequence analysis. The experiments outlined in this application build on the most exciting discoveries made during the previous funding period; charting highly significant correlations between alternative splicing and cis-acting RNA splicing elements, the discovery of a mechanism that describes position-dependent activities of splicing regulators, and the discovery that the retention of spliceosomal components along ligated exons ensures efficient processing. The proposal has three major goals: 1) To investigate experimentally and computationally the complexity of exon selection. A systems approach is proposed to examine methodically how the combination of cis-acting RNA splicing elements influences splice site selection with the objective to generate a splicing code that permits alternative splicing predictions based on sequence analysis. 2) To test the hypothesis that position dependent splicing activation or repression is due to unique interactions between splicing regulatory complexes and spliceosomal components. 3) To determine the mechanisms that ensure highly efficiency processing of multi-intron containing pre-mRNAs. Because defects in splicing lead to many human genetic diseases, including a number of genes that have been implicated in cancer, the proposed research will greatly enhance our understanding of regulated gene expression and human disease. In particular, the results from these studies will provide a quantitative framework for exon recognition and they will permit splicing predictions to classify exonic mutations, thus paving the way for the design of alternative therapeutic approaches to combat disease. PUBLIC HEALTH RELEVANCE: Alternative pre-mRNA splicing is considered one of the most efficient and widespread avenues to generate multiple protein isoforms from individual genes. While many different cis-acting RNA splicing elements have been shown to influence alternative splicing, it is currently unknown how they influence each other mediate exon inclusion or exclusion. In this application we propose to use a systems approach to define a splicing code that predicts the probability of exons to be recognized by the spliceosome and to determine the mechanisms that ensure efficient processing of multi-intron pre-mRNAs.